Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells.

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent.